1. Field of the Invention
Cloning the DNA sequence that corresponds to a genomic defect has been essential to our understanding of the genetic cause of a disease. Identifying the mRNA species that are specific for a given tissue or for a specific event has been a foundation for many areas of modern biomedical research. If a unique sequence is present, or a common sequence is missed in a tumor tissue when it is compared with its normal counterpart, it can be used as a tumor marker. Finding unique DNA fragments in infectious tissue may help to identify infectious agents (Chang, Y., et al., 1994). To achieve these important goals, developing an efficient methodology to identify a sequence that is uniquely present in one sample in comparing with another has been a central issue.
2. Description of the Related Art
Although numerous methods that are designed to identify the differences in sequences have been reported (Davis 1984; Duguin et al., 1990; Hara et al., 1991; Hendrick et al., 1984; Kunkel et al., 1985; Lamar et al., 1984; Nussbaum, et al., 1987; Sargent et al., 1983), many of those methodologies involved physical separation between testers and drivers, such as hydroxyapatite chromatography (Timblin et al., 1990), a streptavidin-biotin interaction (Wang et al., 1991) or oligo(dT)-latex affinity chromatography (Hara et al., 1993). Generally speaking, these methods are time consuming and non-reproducible.
In the past 12 years, a method, known as polymerase chain reaction (PCR), was described (Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159). It is based on repeat cycling of denaturating the double-stranded DNA, oligonucleotide primer annealing to the DNA template, and followed by primer extension with a thermo-stable DNA polymerase. The PCR amplification process results in the exponential increase of a DNA fragment whose length is limited by the 5' ends of the oligonucleotide primers. Application of PCR to isolate and to analyze a particular DNA region requires knowledge of the DNA sequences flanking the region of interest. This feature generally limits its application to regions of known DNA sequence. In the past 5 years, a PCR-based technique, called representational difference analysis (RDA), employs a representational sampling approach by cutting the DNA into fragments based on its restriction enzyme cutting pattern, and attaching these restriction fragments to a PCR-adapter for PCR amplification. Subsequently, it employs a differential enrichment approach to identify and to enrich the differences between tested DNA samples without physical separation (Lisitsyn et al., 1993; Hubank et al., 1994). However, the protocol for this method was found very complicated and time consuming. It has been difficult to employ this technique for routine studies. The mRNA differential display (Liang et al., 1992) and RNA finger printing (Welsh et al., 1992) by randomly primed PCR on cDNA represent potentially faster and easier techniques to identify differential expression genes. However, high background and false positive results are frequently associated with these methods. These techniques also tend to bias toward those abundantly expressed sequences. Recently, a new PCR based cDNA subtraction technique, termed suppression subtractive hybridization (SSH), was described (Diatchenko et al., 1996). This technique used suppression PCR to preferentially amplify differential tester sequences to generate a cDNA probe library. Although this technique can dramatically enrich some differential DNA fragments, only one cycle of hybridization is permitted. Understandably, significant background may be present.
In view of the problems and limitations associated with the methods discussed above, there remains a strong need for a method with enhanced specificity, sensitivity, and efficiency of identifying the differences of DNA sequences between two samples. In this patent application, I describe a novel polymerase chain reaction (PCR)-reversal subtractive hybridization method that rapidly isolates unique DNA sequences present between two tissues or cell types while employing no physical separation between testers and drivers. This method, referred to as Differential Subtraction Chain (DSC), employs a "negative amplification" strategy to identify, and to enrich the differences between two populations of DNA. This strategy produces fast and efficient isolation of unique tester sequences with minimal background.